Modulation of calcium channels in human erythroblasts by erythropoietin

Erythropoietin (Epo) induces a dose-dependent increase in intracellular free Ca²⁺ ([Ca²⁺](i)) in human erythroblasts, which is dependent on extracellular Ca²⁺ and blocked by high doses of nifedipine or Ni²⁺. In addition, pretreatment of human erythroblasts with mouse antihuman erythropoietin receptor antibody but not mouse immunopure IgG blocked the Epo-induced [Ca²⁺], increase, indicating the specificity of the Ca²⁺ response to Epo stimulation. In this study, the erythropoietin-regulated calcium channel was identified by single channel recordings. Use of conventional whole cell patch-clamp failed to detect Epo-induced whole cell Ca²⁺ current. To minimize washout of cytosolic constituents, we next used nystatin perforated patch, but did not find any Epo-induced whole cell Ca²⁺ current. Using Ba²⁺ (30 mmol/L) as charge carrier in cell-attached patches, we detected single channels with unitary conductance of 3.2 pS, reversal potential of +72 mV, and whose unitary current (at +10 mV) increased monotonically with increasing Ba²⁺ concentrations. Channel open probability did not appreciably change over the voltage range (-50 to +30 mV) tested. Epo (2 U/mL) increased both mean open time (from 4.27 ± 0.75 to 11.15 ± 1.80 ms) and open probability (from 0.26 ± 0.06 to 2.56 ± 0.59%) of this Ba²⁺permeable channel. Our data strongly support the conclusion that the Epo-induced [Ca²⁺](i) increase in human erythroblasts is mediated via Ca²⁺ entry through a voltage-independent Ca²⁺ channel.